CN103642890A - Method for preparing ethyl alcohol by adopting carrier fermenting technique - Google Patents

Abstract

The invention discloses a method for preparing ethyl alcohol by adopting a carrier fermenting technique. The method comprises the following steps of: respectively culturing saccharomyces cerevisiae and aspergillus niger; inoculating the saccharomyces cerevisiae into the culture solution of the aspergillus niger and culturing to form mixed mycelium pellets; and pouring the mixed mycelium pellets into a fermentation medium and carrying out simultaneous saccharification and fermentation at the temperature of 30 DEG C and at the stirring speed of 40-60 revolutions per minute to prepare the ethyl alcohol. According to the invention, the simultaneous saccharification and fermentation process and the microorganism immobilization technique are ingeniously combined, so that the method has the advantages of reducing the viscosity of fermentation liquor, effectively preventing the bacterial contamination, reducing the COD (Chemical Oxygen Demand) value of waste grains liquid and increasing the yield of the ethyl alcohol.

Description

Method for preparing ethanol by carrier fermentation technology

technical field

The invention relates to a method for preparing ethanol by carrier fermentation technology, in particular to a method for fermenting ethanol by organically combining immobilized carrier as a fermentation strain with synchronous saccharification and fermentation technology.

Background technique

Fuel ethanol was first fermented from corn, but this process uses a large amount of corn, which poses a threat to national food security. Since 2006, my country has issued a series of policies, emphasizing that the development of fuel ethanol should adhere to the "non-grain" principle, and control the blind development of the deep processing industry of corn products. The main ingredient of Jerusalem artichoke (commonly known as Jerusalem artichoke, Jiangbula, and ghost ginger) is inulin, which is the simplest type of fructan, with a sugar content of 60% to 70% of its dry weight. The hydrolysis of Jerusalem artichoke is easier than that of starchy raw materials. It does not require high temperature liquefaction. It can be directly hydrolyzed with inulinase to obtain fermentable sugar. The process is simple and the energy consumption is low. Compared with vegetarian biomass, it is easier to realize industrialization. Compared with other crops, Jerusalem artichoke has many obvious advantages, such as strong adaptability, resistance to barrenness, cold resistance, drought resistance, easy planting, and high yield. It can be planted and grown in deserts, saline-alkali lands or tidal flats, and can be irrigated by non-irrigation water resources of marine aquaculture wastewater. Jerusalem artichoke is a potential raw material for ethanol fermentation. Compared with starchy raw materials, Jerusalem artichoke can be gelatinized at low temperature, which has advantages in energy saving. In 2007, the National Development and Reform Commission's "10th Five-Year Plan for Bioindustry Development" forwarded by the General Office of the State Council clearly stated that "support the production of fuel ethanol from non-grain raw materials such as sweet sorghum, cassava and Jerusalem artichoke", and encourage non-grain raw material fuel ethanol Technology development and industrial development.

The bacteria used in the bio-fermentation of ethanol from Jerusalem artichoke are generally yeasts. Because Jerusalem artichoke cannot be directly used, it needs to be saccharified first, so the fermentation methods include simultaneous saccharification and fermentation and first saccharification and then fermentation. The so-called simultaneous saccharification and fermentation refers to saccharification and fermentation. Mixed cultivation of fermentation strains, fermentation while saccharification, first saccharification followed by fermentation refers to the use of saccharification strains to hydrolyze inulin, and then use fermentation strains to ferment. The advantage of synchronous saccharification and fermentation is that the fermentable sugar hydrolyzed in the fermented mash is fermented into ethanol in time without accumulation in the process, preventing the accumulation of sugar and inhibiting the secretion of inulinase, avoiding the phenomenon of substrate inhibition, and effectively preventing impurities Bacterial pollution, improve the yield of ethanol, but the current domestic technology level of alcoholic fermentation technology using Jerusalem artichoke as raw material is low, the utilization rate of inulin is not high, and the loss is relatively large. In addition, free strains have the problem that it is difficult to recycle strains, resulting in high production costs and environmental pollution. Immobilized cell technology can solve this problem well. The so-called immobilized cell technology refers to the biological Cells are fixed in a certain way and used as a solid biocatalyst. In ethanol production, immobilizing yeast will greatly improve the efficiency of enzymatic reaction, so it has a good application prospect. However, the currently disclosed yeast immobilization technology has high cost and single immobilization technology.

Chang Baolei from Dalian University of Technology studied the method of producing ethanol from Jerusalem artichoke in his master's thesis, which uses flocculation yeast as the fermentation strain to prepare ethanol. The disadvantage of this method is that it has high requirements for the strain. The saccharification ability must also have the fermentation ability, so the substrate utilization efficiency and fermentation ability of the strain will be affected to a certain extent, which will affect the yield of ethanol.

Ge Xiangyang et al. (Ge Xiangyang, Zhang Weiguo, Breeding of Aspergillus niger strains in simultaneous saccharification and fermentation of Jerusalem artichoke to produce alcohol, Journal of Food and Biotechnology, Vol. method to produce ethanol. The disadvantage of this method is that the strains are not immobilized, high-density fermentation cannot be carried out, which affects the utilization rate of the equipment; the fermentation strains are not recovered, resulting in waste of fermentation strains and easy to cause environmental pollution.

Contents of the invention

Aiming at the deficiencies in the prior art, the present invention provides a method for preparing ethanol by carrier fermentation technology, which combines and improves carrier-immobilized strains as fermentation strains and synchronous saccharification and fermentation technology, which simplifies the process route, The yield of ethanol is improved and the environmental pollution is reduced.

The carrier fermentation technology refers to the method of immobilizing fermentation strains with a microbial carrier, and using the carrier-immobilized strains to ferment and produce ethanol. At present, there is no technology disclosure that combines carrier fermentation technology with simultaneous saccharification and fermentation technology for ethanol production. Generally, these two technologies are used alone; there is no relevant report on the use of immobilized carriers as fermentation strains. The present invention proposes the concept and technical operation method of using carrier-immobilized strains as fermentation strains for the first time. In the present invention, on the basis of adopting inulinase-producing Aspergillus niger and Saccharomyces cerevisiae as strains to prepare ethanol by synchronous saccharification and fermentation technology, after creative research, according to the characteristic that Jerusalem artichoke needs to be saccharified first and then fermented, Aspergillus niger and Saccharomyces cerevisiae Through mixed culture, a strain mixture of Aspergillus niger encapsulating Saccharomyces cerevisiae was formed. The strain mixture encapsulated Saccharomyces cerevisiae in Aspergillus niger, which not only realized the immobilization of Saccharomyces cerevisiae, but also realized simultaneous saccharification, saving cell immobilization The cost is reduced, the process is simplified, the tolerance of yeast cells to ethanol is improved, the viscosity of the fermentation broth is reduced, the COD of the waste liquid is reduced, and the yield of ethanol is increased.

Concrete technical scheme of the present invention is as follows:

A method for preparing ethanol by carrier fermentation technology is characterized in that it comprises the following steps:

(1) Inoculate Saccharomyces cerevisiae into the seed medium according to the inoculum amount of 5-8%, culture at 30°C and 100r/min for 30 hours, collect the Saccharomyces cerevisiae cells after centrifuging the obtained fermentation broth;

(2) Inoculate the spore suspension of Aspergillus niger into the enzyme production medium according to the inoculation amount of 2-5%, and cultivate it at 30°C and 40r/min until the OD ₆₀₀ is 0.3-0.5 to obtain the Aspergillus niger culture solution ;

(3) Insert the yeast cells collected in step (1) into the culture medium of Aspergillus niger in step (2), so that the number ratio of Aspergillus niger and Saccharomyces cerevisiae is 1:3-10, supplement with Jerusalem artichoke powder, The concentration of peptone and (NH ₄ ) ₂ SO ₄ is 25g/L, 10g/L and 5g/L respectively, and then mixed culture at 30°C and 100-200r/min until the _OD600 is 0.6-1, Realize the encapsulation of Aspergillus niger on Saccharomyces cerevisiae to obtain mixed mycelium balls;

(4) Filter the mixed mycelium balls prepared in step (3) and pour them into the fermentation medium according to the inoculation amount of 10%, and synchronously saccharify and ferment to prepare ethanol under the conditions of 30°C and 40-60r/min.

On the basis of the above-mentioned method of using Aspergillus niger to wrap the mixed mycelium balls of Saccharomyces cerevisiae for synchronous saccharification and fermentation of ethanol, the present invention further carried out a large number of experiments and optimized each process condition, thereby ensuring further improvement of fermentation results. details as follows:

In step (4), after the fermentation is completed, the mixed mycelium balls are filtered out, washed with distilled water, and then added to a new fermentation medium for fermentation to prepare ethanol. The strain mixture can be recycled 25-30 times. The strains can be recycled, which greatly reduces the production cost.

In step (1), the composition of the seed medium is preferably: yeast extract 10 g/L, peptone 20 g/L, glucose 20 g/L, water balance, and natural pH.

In step (2), the composition of the enzyme production medium is preferably: Jerusalem artichoke powder 25g/L, (NH ₄ ) ₂ SO ₄ 5g/L, peptone 10g/L, KH ₂ PO ₄ 6 g/L, NaCl 5 g/L , MgSO ₄ ·7H ₂ O 0.5g/L, FeSO ₄ ·7H ₂ O 0.001 g/L, pH 5, 0.1Mpa sterilization for 15min.

In step (2), the number of Aspergillus niger in the spore suspension of Aspergillus niger is 10 ⁶ - 10 ⁷ /ml.

In step (3), the number ratio of Aspergillus niger to Saccharomyces cerevisiae is preferably 1:6-10.

In step (3), while supplementing Jerusalem artichoke powder, peptone and (NH ₄ ) ₂ SO ₄ , it is preferable to add mannitol, sodium selenite and boric acid components to the culture medium so that their concentrations are respectively 8g/L , 0.3g/L and 0.1g/L. The mycelium balls formed in this way have better fermentation performance.

In step (4), the composition of the fermentation medium is preferably: Jerusalem artichoke powder 30-40g/L, (NH ₄ ) ₂ SO ₄ 4-6g/L, corn steep liquor 4-6g/L, KH ₂ PO ₄ 5-10g/L L, MgSO ₄ ·7H ₂ O 0.3-0.6g/L, FeSO ₄ ·7H ₂ O 0.001-0.003 g/L, pH 4.5, 0.1Mpa sterilization for 15min. Further, the composition of the fermentation medium is most preferably: Jerusalem artichoke powder 35g/L, (NH ₄ ) ₂ SO ₄ 5g/L, corn steep liquor 5g/L, KH ₂ PO ₄ 6 g/L, MgSO ₄ ·7H ₂ O 0.5 g/L, FeSO ₄ ·7H ₂ O 0.001 g/L, pH 4.5, 0.1Mpa sterilization for 15min.

After adopting the process of the invention, the fermentation time can be shortened to 48h.

The present invention cleverly combines the simultaneous saccharification and fermentation process with the immobilization technology of microorganisms. According to the characteristics of the strain itself, it does not need to introduce additional carriers to immobilize the strains. This technological idea has not been reported at home and abroad. The invention is proposed for the first time and has the following advantages:

1. The present invention produces enzyme while saccharifying, so that the fermentable sugar hydrolyzed in the fermented mash is fermented into ethanol in time, does not accumulate in the process, prevents sugar from accumulating and inhibits the secretion of inulinase, and avoids the phenomenon of substrate inhibition. It can effectively prevent the contamination of bacteria and increase the yield of ethanol;

2. The present invention adopts mycelium balls as the immobilization means, which reduces the immobilization cost and the viscosity of the fermented liquid. In the process of ethanol fermentation of Jerusalem artichoke, due to water absorption and swelling of dry Jerusalem artichoke, when the substrate concentration increases, the viscosity of the feed liquid increases, flow and mixing are difficult, affecting mass transfer and heat transfer, and the concentration of solids becomes the limiting factor of fermentation. High-viscosity feed liquid affects the heat transfer and mass transfer effect in the fermentation process, and the CO ₂ produced in the fermentation process will also accumulate due to poor discharge, resulting in slow fermentation speed, long fermentation time (more than 60 h), incomplete reaction, and ethanol Yield decreased significantly. However, in the present invention, strains are specially cultivated to produce mixed mycelium balls. The shape of the mycelium balls makes the flow resistance much smaller than that of cells, which can significantly reduce the viscosity of the fermentation broth and increase the yield of ethanol.

3. The present invention collects the fermentation strains to form mycelium balls instead of free strains, which can reach a higher cell concentration in the fermenter, greatly improving the fermentation rate of the ethanol fermentation process, and correspondingly improving the fermentation rate. The equipment production intensity of the tank is reduced, and the total volume scale of the equipment and the investment in the construction of fixed assets of the fermentation tank are reduced.

4. The present invention uses Aspergillus niger to immobilize Saccharomyces cerevisiae without additional carriers, saving the cost of cell immobilization, Saccharomyces cerevisiae is easy to recover from the fermentation system, has a long service life and can be used repeatedly, convenient and extensive operation, and low cost , easy to realize industrial production;

5. Due to the formation of mycelial balls by wrapping Saccharomyces cerevisiae in Aspergillus niger, the mycelia of Aspergillus niger is rich in lipoproteins that can improve yeast vitality and alcohol resistance, which changes the microenvironment of Saccharomyces cerevisiae cells after immobilization, making wine Yeast cells have increased tolerance to ethanol.

6. After Saccharomyces cerevisiae is completely immobilized in Aspergillus niger, it can be recycled, and the COD of the waste liquid produced after the alcohol rectification of the fermentation liquid is reduced from 50000-60000ppm in the existing alcohol fermentation process to 20000-30000ppm, which is alcohol The fermentation industry has created favorable conditions for reducing waste at the source of pollutants and clean production.

Detailed ways

The present invention will be further described below through specific examples. It should be understood that the following descriptions are only for explaining the present invention and not limiting it.

The Aspergillus niger (CMCC98003) used in the present invention was purchased from Nanjing Bianzhi Instrument Equipment Co., Ltd., and the Saccharomyces cerevisiae used was purchased from Angel Yeast Co., Ltd.

Example 1

A method for producing ethanol through synchronous saccharification and fermentation of mycelium as carrier and fermentation strain:

1. Inoculate the Saccharomyces cerevisiae strain into a 250ml Erlenmeyer flask containing 100ml seed medium according to the inoculum amount of 5%, culture it in a shaker at 30°C and 100r/min for 30 hours, and centrifuge the fermentation broth at 5000r/min for 5min. Yeast cells were collected; the seed medium was yeast extract 10 g/L, peptone 20 g/L, glucose 20 g/L, and natural pH.

2. Make Aspergillus niger into a spore suspension with sterile water, inoculate it into a 250ml Erlenmeyer flask containing 100ml of enzyme-producing medium according to the inoculum amount of 4%, and cultivate it in a shaker at 30°C and 40r/min for 18h, and the spores The OD ₆₀₀ value of the expanded culture of the suspension was 0.4. Among them, the formulation of the enzyme production medium is Jerusalem artichoke powder 25g/L, (NH ₄ ) ₂ SO ₄ 5g/L, peptone 10g/L, KH ₂ PO ₄ 6 g/L, NaCl 5 g/L, MgSO ₄ 7H ₂ O 0.5g/L, FeSO ₄ 7H ₂ O 0.001 g/L, pH 5, 0.1Mpa sterilization for 15min;

3. Inoculate the yeast cells into the Aspergillus niger expanded culture medium in step 2, so that the number ratio of Aspergillus niger and Saccharomyces cerevisiae is 1:6, and then supplement Jerusalem artichoke powder, peptone and (NH ₄ ) ₂ SO ₄ culture Base components to 25g/L, 10g/L and 5g/L, cultured in a shaker at 30°C and 140r/min for 30 h, during the formation of Aspergillus niger mycelium balls, wrapped Saccharomyces cerevisiae inside, and obtained embedding The _OD600 value of the mixed mycelium ball with Saccharomyces cerevisiae was 0.6;

4. After filtering the prepared mixed mycelium balls, pour them into the fermentation medium according to the inoculation amount of 10%, and carry out synchronous saccharification and fermentation in a shaker at 30°C and 40r/min to produce ethanol; In the bottle, the composition of the fermentation medium is: Jerusalem artichoke powder 35g/L, (NH ₄ ) ₂ SO ₄ 5g/L, corn steep liquor 5g/L, KH ₂ PO ₄ 6 g/L, MgSO ₄ ·7H ₂ O 0.5g/ L, FeSO ₄ 7H ₂ O 0.001 g/L, pH 4.5, sterilized at 0.1Mpa for 15 minutes;

5. Fermentation ends after 48 hours, and the mixed mycelium balls are recovered by filtration, washed with distilled water, and then step 4 is repeated 25 times, and the mixed mycelium balls are used for ethanol fermentation. After the fermentation, the ethanol concentration reaches 12.48 g/L, and the ethanol gets The yield (the amount of ethanol produced per gram of Jerusalem artichoke flour) was 0.357g/g.

Example 2

Ethanol was prepared by the following method:

1. Inoculate the Saccharomyces cerevisiae strain into a 250ml Erlenmeyer flask containing 100ml seed medium according to the inoculum amount of 8%, culture it in a shaker at 30°C and 100r/min for 30 hours, and centrifuge the fermentation broth at 5000r/min for 5min. Yeast cells were collected; the seed medium was yeast extract 10 g/L, peptone 20 g/L, glucose 20 g/L, and natural pH.

2. Make Aspergillus niger into a spore suspension with sterile water, inoculate it into a 250ml Erlenmeyer flask containing 100ml of enzyme-producing medium according to the inoculation amount of 2%, and cultivate it in a shaker at 30°C and 40r/min for 18h, and the spores The OD ₆₀₀ value of the expanded culture of the suspension was 0.5. Among them, the formulation of the enzyme production medium is Jerusalem artichoke powder 25g/L, (NH ₄ ) ₂ SO ₄ 5g/L, peptone 10g/L, KH ₂ PO ₄ 6 g/L, NaCl 5 g/L, MgSO ₄ 7H ₂ O 0.5g/L, FeSO ₄ 7H ₂ O 0.001 g/L, pH 5, 0.1Mpa sterilization for 15min;

3. Inoculate the yeast cells into the Aspergillus niger expanded culture medium in step 2, so that the number ratio of Aspergillus niger and Saccharomyces cerevisiae is 1:10, and then supplement Jerusalem artichoke powder, peptone and (NH ₄ ) ₂ SO ₄ culture Base components to 25g/L, 10g/L and 5g/L, 30°C, 180r/min shaker culture for 30 hours, during the formation of Aspergillus niger mycelium balls, Saccharomyces cerevisiae was wrapped inside to obtain embedding The _OD600 value of the mixed mycelial ball with Saccharomyces cerevisiae was 0.8;

4. After filtering the prepared mixed mycelium balls, pour them into the fermentation medium according to the inoculation amount of 10%, and carry out synchronous saccharification and fermentation in a shaker at 30°C and 60r/min to produce ethanol; In the bottle, the composition of the fermentation medium is: Jerusalem artichoke powder 35g/L, (NH ₄ ) ₂ SO ₄ 5g/L, corn steep liquor 5g/L, KH ₂ PO ₄ 6 g/L, MgSO ₄ ·7H ₂ O 0.5g/ L, FeSO ₄ 7H ₂ O 0.001 g/L, pH 4.5, sterilized at 0.1Mpa for 15 minutes;

5. After 48 hours, the fermentation ends, the mixed mycelium balls are recovered by filtration, washed with distilled water, and then step 4 is repeated 28 times, and the mixed mycelial balls are used for ethanol fermentation. After the fermentation, the ethanol concentration reaches 13.12 g/L, and the ethanol gets The rate is 0.375g/g.

Example 3

Study on the influence of the formation of mixed mycelium balls on the fermentation effect under different rotational speed conditions

Ethanol is prepared by fermentation according to the method of Example 1, the difference is: in step 3, when Aspergillus niger and Saccharomyces cerevisiae are mixed cultured, the shaker speed is mixed according to 80r/min, 100r/min, 180r/min, and the final obtained ethanol The performance parameters are as follows.

Through the comparison of Example 1 and the above data, it can be seen that the rotating speed of the shaking table has a great influence on the formation of mixed mycelium balls, the yield of ethanol, and the like.

Example 4

Study the effect of different strain mixing ratios on the fermentation effect

Ethanol is prepared by fermentation according to the method of Example 1, the difference is: in step 3, when Aspergillus niger and Saccharomyces cerevisiae are mixed cultured, according to the number ratio of Aspergillus niger and Saccharomyces cerevisiae respectively 1:1, 1:3, 1:6, 1:10, 1:12 for mixed culture, the performance parameters of the final obtained ethanol are as follows.

By comparing Example 1 with the above data, it can be seen that the formation of mixed mycelium balls has requirements on the ratio of the two bacteria, and the mixing ratio is too low or too high to affect the fermentation effect of mixed mycelium balls, thereby affecting to the final ethanol concentration.

Example 5

Study on the Effect of Different Mixed Mycelia Culture Medium on Fermentation Effect

Fermentative preparation of ethanol according to the method of Example 1, the difference is: in step 3, the yeast cells are inoculated into the Aspergillus niger expansion culture solution of step 2, so that the number ratio of Aspergillus niger and Saccharomyces cerevisiae is 1:6, Then add Jerusalem artichoke powder, peptone, (NH ₄ ) ₂ SO ₄ , mannitol, sodium selenite and boric acid medium components to the culture medium, so that their concentrations are respectively 25g/L, 10g/L, 5g/L , 8g/L, 0.3g/L and 0.1g/L, cultured in a shaker at 30°C and 140r/min for 30 hours, during the formation of Aspergillus niger mycelium balls, Saccharomyces cerevisiae was wrapped inside to obtain embedding The _OD600 value of the mixed mycelial pellet with S. cerevisiae was 0.6.

The ethanol performance parameter obtained by this method is compared with that of Example 1, and the results are as follows.

Example 6

Study the effect of different fermentation media on the fermentation effect

Ethanol was prepared by fermentation according to the method of Example 1, the difference being that in step 4, the fermentation medium was different, and the fermentation medium used in the first group was: Jerusalem artichoke powder 30g/L, (NH ₄ ) ₂ SO ₄ 6g/L, corn slurry 4g/L, KH ₂ PO ₄ 10g/L, MgSO ₄ 7H ₂ O 0.3g/L, FeSO ₄ 7H ₂ O 0.003 g/L, pH 4.5; the fermentation medium used in the second group was: Jerusalem artichoke powder 35g /L, (NH ₄ ) ₂ SO ₄ 5g/L, corn steep liquor 5g/L, KH ₂ PO ₄ 6 g/L, MgSO ₄ 7H ₂ O 0.5g/L, FeSO ₄ 7H ₂ O 0.001 g/L , pH 4.5; the fermentation medium used in the third group was: Jerusalem artichoke powder 40g/L, (NH ₄ ) ₂ SO ₄ 4g/L, corn steep liquor 6g/L, KH ₂ PO ₄ 5 g/L, MgSO ₄ ·7H ₂ O 0.6 g/L, FeSO ₄ ·7H ₂ O 0.002 g/L, pH 4.5.

The performance parameters of the final obtained ethanol are shown in the following table.

Claims (10)

1. carrier fermentation technique is prepared a method for ethanol, it is characterized in that comprising the following steps:

(1) yeast saccharomyces cerevisiae is inoculated in seed culture medium according to the inoculum size of 5-8%, under 30 ℃, the condition of 100r/min, cultivates 30 h, the centrifugal rear collection brewing yeast cell of gained fermented liquid;

(2) spore suspension of aspergillus niger is inoculated in culture medium according to the inoculum size of 2-5%, under 30 ℃, the condition of 40r/min, is cultured to OD ₆₀₀for 0.3-0.5, obtain aspergillus niger nutrient solution;

(3) yeast cell of step (1) being collected is linked in the aspergillus niger nutrient solution of step (2), makes the bacterial classification number of aspergillus niger and yeast saccharomyces cerevisiae than being 1:3～10, supplements jerusalem artichoke powder, peptone and (NH ₄) ₂sO ₄make their concentration be respectively 25g/L, 10g/L and 5g/L, then at 30 ℃, under the condition of 100～200r/min, mixed culture is to OD ₆₀₀for 0.6-1, realize the parcel of aspergillus niger to yeast saccharomyces cerevisiae, obtain mixed bacterium pompon;

(4) the mixed bacterium pompon that step (3) made is poured in fermention medium according to 10% inoculum size after filtering, and under 30 ℃, the condition of 40-60r/min, simultaneous saccharification and fermentation is prepared ethanol.

2. method according to claim 1, is characterized in that: in step (4), after fermentation ends, mixed bacterium pompon is filtered out, after distilled water cleans, rejoin in new fermention medium and carry out alcohol prepared by fermenting, bacterial classification mixture can recirculation be used 25-30 time.

3. method according to claim 1, is characterized in that: in step (1), seed culture medium consists of: yeast extract paste 10 g/L, peptone 20 g/L, glucose 20 g/L, water surplus, natural pH.

4. method according to claim 1, is characterized in that: in step (2), culture medium consists of: jerusalem artichoke powder 25g/L, (NH ₄) ₂sO ₄5g/L, peptone 10g/L, KH ₂pO ₄6 g/L, NaCl 5 g/L, MgSO ₄7H ₂o 0.5g/L, FeSO ₄7H ₂o 0.001 g/L, pH 5,0.1Mpa sterilizing 15min.

5. according to the method described in claim 1 or 4, it is characterized in that: in step (2), in the spore suspension of aspergillus niger, the number of aspergillus niger is 10 ^6-10 ⁷individual/ml.

6. method according to claim 1, is characterized in that: in step (4), fermention medium consists of: jerusalem artichoke powder 30-40g/L, (NH ₄) ₂sO ₄4-6g/L, corn steep liquor 4-6g/L, KH ₂pO ₄5-10g/L, MgSO ₄7H ₂o 0.3-0.6g/L, FeSO ₄7H ₂o 0.001-0.003 g/L, pH 4.5,0.1Mpa sterilizing 15min.

7. method according to claim 6, is characterized in that: in step (4), fermention medium consists of: jerusalem artichoke powder 35g/L, (NH ₄) ₂sO ₄5g/L, corn steep liquor 5g/L, KH ₂p O ₄6 g/L, MgSO ₄7H ₂o 0.5g/L, FeSO ₄7H ₂o 0.001 g/L, pH 4.5,0.1Mpa sterilizing 15min.

8. according to the method described in claim 1,6 or 7, it is characterized in that: in step (4), fermentation time is 48h.

9. according to the method described in any one in claim 1-4,6 or 7, it is characterized in that: in step (3), supplement jerusalem artichoke powder, peptone and (NH ₄) ₂sO ₄time, in nutrient solution, add N.F,USP MANNITOL, Sodium Selenite and boric acid, make their concentration be respectively 8g/L, 0.3g/L and 0.1g/L.

10. method according to claim 1, is characterized in that: in step (3), the bacterial classification number of aspergillus niger and yeast saccharomyces cerevisiae is than being 1:6～10.